The ways by which gene expression is altered without any change in the underlying DNA sequence is referred to as epigenetics. We envision that the secondary structure of DNA such as the G-quadruplex (GQ) may be yet another epigenetic element that contributes to gene regulation. Once regarded as an experimental artifact, GQ has been rediscovered through recent studies that clearly demonstrated the presence of GQ in the human genome and its enrichment in important regulatory regions such as near gene promoters and replication origin. In addition, there is an accumulating evidence that the GQ elements control gene expression at the level of transcription and translation. These findings are leading to an emerging hypothesis that the GQ in genomic DNA may act like a switch to turn the gene expression on and off. One of the big challenges in testing this hypothesis lies at the high level of heterogeneity in various GQ forming sequences. It is unrealistic to rely on the previously published results since majority of studies focused on probing GQ in the context of single strand DNA (ssDNA) when the genomic GQs are expected to form in double strand DNA (dsDNA). We have devised biochemical and biophysical platforms for examining various GQ forming sequences in dsDNA. Our recently published data already suggest that the GQ formation is drastically different between the ssDNA and dsDNA. We present our preliminary data on GQ folding propensity analysis on 438 GQ forming sequences. Based on this mapping, we propose to examine more complex GQ sequences that are pertinent to/represented in genomic DNA (Aim 1). We present our result on the DNA and RNA polymerase assays which measure strength of GQ sequences and the corresponding barrier effect. With a newly established real time assays which detect mRNA production and GQ formation in mRNA, we will continue to examine the GQ effect in blocking DNAP and RNAP as a way of estimating their role in the process of replication and transcription (Aim 2). The same GQ sequences tested above were cloned into plasmid and E. coli chromosome to measure the magnitude of gene activation/repression induced by the GQ elements. Our new preliminary data demonstrates that the gene repression effect is dependent on GQ folding stability and the GQ positioned in non-template DNA. This work will continue and will be followed by mammalian cell testing (Aim 3). Using these platforms, we propose to test three possible modes of gene switching that includes (i) rheostat (gradient) in which different strength of GQ folding lead to variable levels of gene repression/activation, (ii) rotary (stepwise) where there are several distinct levels of GQ switches that control the gene activity, (iii) toggle (binary, on-off) switch which simply has a set threshold GQ strength beyond which gene expression is turned on. Our systematic approach and stepwise analysis will provide quantitative understanding about the GQ folding strength and its connection to biological processes including replication, transcription and translation.